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J Biol Chem, Vol. 275, Issue 18, 13441-13447, May 5, 2000

Zn²⁺ Inhibits -Ketoglutarate-stimulated Mitochondrial Respiration and the Isolated -Ketoglutarate Dehydrogenase Complex^*

Abraham M. Brown^§¶, Bruce S. Kristal^§, Michelle S. Effron, Alexander I. Shestopalov, Paul A. Ullucci^**, K.-F. Rex Sheu, John P. Blass, and Arthur J. L. Cooper^§

From the  Burke Medical Research Institute, White Plains, New York 10605, the Departments of ^§ Biochemistry,  Neurology and Neuroscience, and  Medicine, Weill Medical College of Cornell University, New York, New York 10021 and ^** ESA, Inc., Chelmsford, Massachusetts 01824

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Intracellular free Zn²⁺ is elevated in a variety of pathological conditions, including ischemia-reperfusion injury and Alzheimer's disease. Impairment of mitochondrial respiration is also associated with these pathological conditions. To test whether elevated Zn²⁺ and impaired respiration might be linked, respiration of isolated rat liver mitochondria was measured after addition of Zn²⁺. Zn²⁺ inhibition (K_i^app = ~1 µM) was observed for respiration stimulated by -ketoglutarate at concentrations well within the range of intracellular Zn²⁺ reported for cultured hepatocytes. The bc₁ complex is inhibited by Zn²⁺ (Link, T. A., and von Jagow, G. (1995) J. Biol. Chem. 270, 25001-25006). However, respiration stimulated by succinate (K_i^app = ~6 µM) was less sensitive to Zn²⁺, indicating the existence of a mitochondrial target for Zn²⁺ upstream from bc₁ complex. Purified pig heart -ketoglutarate dehydrogenase complex was strongly inhibited by Zn²⁺ (K_i^app = 0.37 ± 0.05 µM). Glutamate dehydrogenase was more resistant (K_i^app = 6 µM), malate dehydrogenase was unaffected, and succinate dehydrogenase was stimulated by Zn²⁺. Zn²⁺ inhibition of -ketoglutarate dehydrogenase complex required enzyme cycling and was reversed by EDTA. Reversibility was inversely related to the duration of exposure and the concentration of Zn²⁺. Physiological free Zn²⁺ may modulate hepatic mitochondrial respiration by reversible inhibition of the -ketoglutarate dehydrogenase complex. In contrast, extreme or chronic elevation of intracellular Zn²⁺ could contribute to persistent reductions in mitochondrial respiration that have been observed in Zn²⁺-rich diseased tissues.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The pool of cellular Zn²⁺ that is not tightly bound to macromolecules or to other ligands and can be readily chelated by Zn²⁺-sensitive chromophores or fluorophores has been termed "chelatable zinc" (1). Interest in the biological function of chelatable Zn²⁺ has grown steadily during the last decade. This interest was stimulated in part by the recognition that the concentration of chelatable Zn²⁺ is elevated in some cerebral regions following episodes of transient ischemia (2, 3) or excitotoxic injury (4). Elevated intracellular Zn²⁺ has also been observed in models of cardiac ischemia and cardiac inflammation (5, 6). Excess intracellular Zn²⁺ is toxic to neurons (7-9). Various mechanisms for the toxic activity of Zn²⁺ have been proposed including modulation of amino acid receptor activity (10-13), alteration of nerve growth factor binding (14), induction of gene expression (15), and alterations of mitochondrial function (7, 8). Elevated intracellular free Zn²⁺ and reduced carbohydrate flux through mitochondrial energy pathways (16-21) are prominent pathological features in some neurological and cardiac diseases.

Oxidative energy metabolism is impaired in many neurodegenerative disorders (22-24). One component of energy metabolism that has been extensively studied is the -ketoglutarate dehydrogenase complex (KGDHC).¹ Reductions in KGDHC activity or protein abundance in brain occur in several neurological diseases (25-30). The cause of this deficit has not been established. Reduced concentration and/or activity of enzymes involved in mitochondrial carbohydrate metabolism has been documented for both chronic ischemia and ischemia-reperfusion injury in heart and brain (20, 21, 31).

Studies on intact mitochondria have revealed that Zn²⁺ inhibits respiration supported by combined glutamate and malate (32) or -hydroxybutyrate (33). Subsequent studies identified complex III, specifically the bc₁ complex, as the site of Zn²⁺ binding and inhibition (34, 35). (Similarly, cytochrome b₅₆₂-o complex of aerobically grown Escherichia coli K12 is inhibited by Zn²⁺ (36).) These studies utilized substrates that enter the respiratory chain downstream from complex I such as succinate, nonylubihydroquinone, or duroquinol (32, 34, 35). The choice of substrates in the earlier studies precluded detection of Zn²⁺ inhibition of upstream dehydrogenases. Evidence of disease-linked impairment of KGDHC activity (see previous paragraph), which is part of the (Krebs) tricarboxylic acid cycle that is upstream from complex I, motivated us to examine the effect of Zn²⁺ on earlier stages of mitochondrial energy metabolism. Because the cytosolic concentration of free Zn²⁺ in dissociated hepatocytes is in the range of 0.6-2.7 µM (37), we tested the effect of submicromolar to micromolar Zn²⁺ upon mitochondrial respiration.

KGDHC converts -ketoglutarate (-KG), coenzyme A (CoA), and NAD⁺ to succinyl-CoA, CO₂, and NADH in the presence of thiamin pyrophosphate (TTP) (38). The complex subunit structure of KGDHC has been extensively studied. The sequential activities catalyzed by KGDHC (39) are summarized below. KGDHC activity is feedback-inhibited by the reaction products NADH and succinyl-CoA (40). KGDHC is also sensitive to metals; activity is enhanced by millimolar Mg²⁺ or micromolar Ca²⁺ (41) but is inhibited by higher (>mM) concentrations of Ca²⁺ (42, 43).

In this paper, we report that micromolar concentrations of Zn²⁺ in the assay buffer inhibit respiration of intact liver mitochondria. Respiration stimulated by -KG is more sensitive to Zn²⁺ inhibition than respiration stimulated by other mitochondrial energy substrates. Experiments with purified enzyme indicated that KGDHC is inhibited by submicromolar concentrations of Zn²⁺. The possible roles of Zn²⁺ in the regulation of mitochondrial respiration and in mitochondrial pathology are discussed.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- Sodium -KG, sodium succinate, L-glutamate, malate, NADH, NAD⁺, TPP, Tris, CoASH, dithiothreitol (DTT), fatty acid-free bovine serum albumin (BSA; A-6003), DL-6,8-thioctic acid amide (LipS₂), 2,6-dichlorophenol-indophenol, p-iodonitrotetrazolium violet, EDTA, ZnCl₂, CaCl₂, and MgCl₂ were purchased from Sigma. Whenever possible, ultrapure grades of reagents were used; all other reagents were of the highest quality available commercially. Stock solutions (100 mM) of lipoamide were prepared in Me₂SO and used within a few hours or stored in frozen aliquots at 20 °C until needed. Where required, lipoamide (i.e. -SS- form) was reduced to dihydrolipoamide (i.e. -(SH)₂ form) in situ by inclusion of 0.1 mM DTT in the reaction solution. Buffer solutions were prepared using water that was distilled and then deionized through alternating anion-cation exchangers (Barnstead), resulting in >15 M/cm resistance. Absolute Zn²⁺ concentrations were determined by flame atomic absorption analysis using a Hitachi 6100 Zeeman Background Correction atomic absorption spectrometer. Analysis of the buffers used in all assays indicated that total Zn²⁺ was below the limit of detection (<0.08 µM).

Enzymes-- Pig heart KGDHC (0.5 unit/mg of protein (11.1 mg/ml) in a solution containing 50% glycerol, 25% sucrose, 5 mM EGTA, 5 mM DTT, 10 mg/ml BSA, 1% Triton X-100, 0.01% sodium azide, and 50 mM potassium phosphate, pH 6.9); pig heart lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; the E3 component of KGDHC) (suspension in 3.2 M ammonium sulfate; 128 units/mg of protein; 11.5 mg/ml)); and pig heart mitochondrial malate dehydrogenase (MDH) (910 units/mg of protein in 50% glycerol containing 50 mM potassium phosphate buffer, pH 7.5, 5.6 mg/ml) were obtained from Sigma. Beef liver glutamate dehydrogenase (GDH) (crystalline suspension in 2 M ammonium sulfate; 20 mg/ml of protein; 120 units/mg) was obtained from Roche Molecular Biochemicals. Enzyme activities are expressed as µmol of product formed per min at 37 °C.

Isolation of Mitochondria-- Rat liver mitochondria were isolated essentially as described previously (44-46). All steps were carried out at 0-4 °C. Four-month-old Fisher 344 male rats were decapitated. Livers were rapidly removed and homogenized with a motor-driven Teflon pestle in buffer A (250 mM mannitol, 75 mM sucrose, 10 mM HEPES, adjusted to pH 7.4 with KOH) supplemented with 100 µM K-EDTA and 500 µM K-EGTA. After centrifugation at 1,000 × g for 10 min, the supernatants were removed and subjected to centrifugation at 10,000 × g for 15 min. The resulting pellets were washed in buffer A supplemented with 100 µM K-EDTA and 500 µM K-EGTA and 0.5% (w/v) fatty acid-free BSA and centrifuged at 10,000 × g, followed by two additional washes in buffer A supplemented with 30 µM EDTA and 0.5% (w/v) fatty acid-free BSA. Following the final wash, mitochondria were resuspended in buffer A and 5 µM K-EDTA. Protein concentrations were estimated spectrophotometrically using BSA as a standard (47).

Mitochondrial Respiration Assays-- Mitochondrial oxygen consumption was measured at 28 °C using a Clark electrode in a computer controlled system (Hansatech, PP Systems, Haverhill, MA) as described previously with minor modification (44). Mitochondria were added to 1.7-ml chambers containing respiration buffer (270 mM sucrose, 10 mM KH₂PO₄, pH 7.4, 3 mM MgCl₂; final mitochondrial concentration, 0.8 ± 0.2 mg protein/ml). Samples were preincubated with or without added ZnCl₂ for 2 min. Following preincubation, state 4 respiration (respiration in the absence of ADP) was initiated by addition of substrate (5 mM -KG, 7.5 mM succinate, or 6.25 mM glutamate/malate). These substrate concentrations were saturating for state 3 respiration under the conditions used (data not shown). After an additional 3 min, state 3 respiration rates (ADP-stimulated) were determined by adding ADP to a final concentration of 0.5 mM (48). Stabilized respiration rates (i.e. 1 min after ADP addition) were determined by least square regression analysis using Hansatech software. The intervals used for determination of the rate of oxygen consumption were 1.5 min for state 4 and 2-3 min for state 3.

Preparation of KGDHC-- The enzyme is sensitive to inhibition by low concentrations of Zn²⁺ (see "Results"). Therefore, as a prerequisite for the study of the effect of Zn²⁺ on KGDHC, the purified enzyme complex was subjected to Sephadex chromatography to remove EGTA, EDTA, and BSA (components of the storage buffer that chelate Zn²⁺ and other divalent ions). An aliquot (100 µl) of the commercial KGDHC preparation was loaded onto a 0.5 × 8 cm Sephadex G-200 (Amersham Pharmacia Biotech) column equilibrated with 50 mM Tris-HCl, pH 7.4, and 0.1 mM DTT. The enzyme was eluted with 100-µl aliquots of the same buffer, and fractions containing enzyme activity (fractions 6-8) were used. Activity of the most-concentrated fractions was stable at 4 °C for 3-7 days.

Enzyme and Subunit Reaction Schemes-- KGDHC is composed of multiple copies of three different subunits that transfer intermediate enzyme products in an ordered fashion (39). The combined E1, E2, and E3 subunits of KGDHC catalyze the following reaction sequence in the presence of -KG, TPP, CoA, and NAD⁺.

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

where [Lip(SH)₂] and [LipS₂] represent the dihydrolipoamide (reduced form) and lipopamide (oxidized form) of the tethered lipoic acid prosthetic group of E2, respectively (39). The overall reaction is as follows.

(Eq. 6)

Assay of the combined E1-E2 activity (Equations 1-3) can be accomplished by introducing free LipS₂ to replace E3-FAD as an electron acceptor (Equation 7). The reduced lipoamide prosthetic group is oxidized by disulfide exchange, allowing E2 to recycle while generating a pool of Lip(SH)₂.

(Eq. 7)

Subsequent quantitation of Lip(SH)₂ is accomplished in an end point assay that measures the burst of NADH produced upon addition of the accumulated Lip[SH]₂ to a mixture of purified E3 and excess NAD⁺.

(Eq. 8)

Reduced E3-FAD then converts NAD⁺ to NADH (Equation 5) giving the following overall reaction.

(Eq. 9)

Assay of E3 activity (independent of E1 and E2) is accomplished by providing exogenous Lip(SH)₂ and NAD⁺ to either KGDHC or isolated E3, as in Equation 9. Reaction 9 is freely reversible. Therefore, the reverse (diaphorase) reaction catalyzed by E3 can be monitored in the presence of NADH and LipS₂ (see below).

Activity Measurements of KGDHC and Its Components-- The volume of the reaction mixtures was 200 µl, except where noted. Activities were determined spectrophotometrically at 23 °C using a 96-well spectrophotometric plate reader (SpectraMax 250, Molecular Dynamics, Sunnyvale, CA). NADH appearance or disappearance was measured at 340 nm (_340 nm = 6.23 × 10³). Typically, the absorbance values of individual wells were determined at 5-s intervals. In all cases where Zn²⁺ was present the cation was added as the chloride salt, prior to initiation of the reaction. Standard assay mixtures were as follows: (i) for KGDHC: 3.2 mM -KG, 2 mM NAD⁺, 0.7 mM TPP, 0.5 mM CoASH, 20 µM CaCl₂, 1 mM MgCl₂, 0.1 mM DTT, and 50 mM Tris-HCl, pH 7.4; (ii) for combined E1 and E2: mixture 1 (100 µl): 4 mM -KG, 0.7 mM TPP, 0.5 mM CoASH, 0.3 mM LipS₂, 20 µM CaCl₂, 1 mM MgCl₂, 50 mM Tris-HCl, pH 7.4, and 12-25 µg/ml desalted KGDHC, and mixture 2 (100 µl): 2 mM NAD⁺, 20 µM EDTA, 10 µl of 1:40 dilution of lipoamide dehydrogenase (E3) suspension, and 50 mM Tris-HCl, pH 7.4. Zn²⁺ was added to mixture 1 before initiating the reaction with -KG. After 15 min, 100 µl of mixture 1 was added to an equal volume of Mixture 2 in a microtiter well. The optical density was recorded before and after mixing. Mixture 1 in the presence of -KG supports the combined E1-E2 activity (Equations 1-3 and 7) generating Lip(SH)₂ as the final product. Addition of mixture 2 promotes the consumption of Lip(SH)₂ and production of NADH (Equation 9). The rapid increase in absorbance, because of reduction of NAD⁺ after combining mixtures 1 and 2, was interpreted as a quantitative measure of the pool of Lip(SH)₂ formed by the combined E1-E2 activities. Note that EDTA was included in mixture 2 to prevent possible inhibition of E3 by chelating Zn²⁺ carried over from Mixture 1; (iii) for E3 forward reaction: 2 mM NAD⁺, 0.25 mM LipS₂, 0.1 mM DTT, and 100 mM Tris-HCl, pH 7.4; and (iv) for E3 reverse reaction: 0.1 mM NADH, 0.1 mM LipS₂, and 100 mM Tris-HCl, pH 7.4.

Measurement of Mitochondrial SDH Activity and Purified GDH and MDH Activities-- For SDH activity, isolated mitochondria (~20 mg/ml protein) were fractured by three freeze-thaw cycles and mixed with an equal volume of reaction buffer (50 mM sodium phosphate, pH 7.4). The standard reaction mixture contained ~0.5 mg/ml mitochondrial protein, 7 mM sodium succinate, 0.5 mM p-iodonitrotetrazolium violet in reaction buffer. Reaction progress was monitored as an increase of absorbance at 490 nm. For GDH, 10 mM -KG, 0.5 mM NADH, 50 mM ammonium sulfate, 0.1 mM ADP, and 100 mM Tris-HCl, pH 7.6, were used. For MDH, 10 mM oxaloacetate (added as a solid immediately before assay), 1 mM NADH, and 100 mM Tris-HCl, pH 7.6, were used.

Data Analyses-- All data are reported as the means ± S.E. Enzyme velocities (V) were determined by regression analysis of the change in NADH absorbance with time. Apparent inhibition constants (K_i^app) for enzyme data were determined by Dixon plots of 1/V plotted against the concentration of added Zn²⁺ (49). K_i^app was the negative of the x intercept calculated from the linear regression coefficients determined using SIGMAPLOT (SPSS, Inc.). Regression analysis for Dixon plots of respiration data was performed using the Robust Regression routine in the NCSS 97 statistics package (Kaysville, UT). Standard errors for K_i^app were calculated from the product of the estimated K_i^app and the estimated confidence value (the root of the sum of the squares of the confidence values for the two regression coefficients). Significance between means was tested using Student's t test (two-tailed).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effect of Zn²⁺ on Mitochondrial Respiration-- Respiration was measured in isolated, intact liver mitochondria by monitoring oxygen consumption using succinate, glutamate/malate, or -KG as substrates. Note that 3 mM Mg²⁺ was included in the respiration buffer to protect mitochondria from induction of the permeability transition (46, 50, 51). Under these conditions Zn²⁺ did not induce mitochondrial swelling, as assessed by absorbance at 540 nm.² Mitochondria were preincubated for 2 min in the presence of 0-6.4 µM Zn²⁺. Substrate was added, and 3 min of state 4 respiration was monitored prior to ADP addition (state 3 respiration). Fig. 1A illustrates that in the presence of succinate state 3 respiration was at least partly maintained at 6.4 µM Zn²⁺. All five mitochondrial preparations tested in the presence of succinate failed to recover state 4 respiration when exposed to 6.4 µM Zn²⁺, which may be due to uncoupling and/or ATPase activation. Glutamate/malate-stimulated state 3 respiration was somewhat inhibited by 0.4 or 1.6 µM Zn²⁺ and completely inhibited by 6.4 µM Zn²⁺ (Fig. 1B). In contrast, partial inhibition of -KG-stimulated State 3 respiration was observed at 0.4 µM, and complete inhibition required only 3.2 µM Zn²⁺ (Fig. 1C). The IC₅₀ values for each substrate (Table I) were statistically different from each other (p < 0.05). The K_i^app for Zn²⁺ inhibition of respiration determined analytically (Fig. 2) also differed significantly for each substrate and was smallest for -KG (Table I).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Selective inhibition of mitochondrial respiration by Zn2+. Oxygen consumption of freshly isolated rat liver mitochondria was monitored as described under "Materials and Methods." Zn2+ concentrations are as indicated. The arrow indicates the addition of ADP to initiate state 3 respiration. Mitochondria were incubated with Zn2+ for 3 min prior to initiation of state 3. A, Respiration in the presence of succinate. B, respiration in the presence of glutamate/malate couple. C, respiration in the presence of -KG.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Analysis of Zn2+ inhibition of mitochondrial respiration. Dixon plots of oxygen consumption by mitochondria in the presence of varying concentrations of Zn2+. The value of V for each point was calculated from the ratio of the experimental respiration rate to the average respiration rate for control samples from the same preparation, which was arbitrarily assigned a value of 1. The different symbols represent four or five independent preparations. A, succinate. B, glutamate/malate. C, -KG.

Effect of Zn²⁺ on SDH, GDH, and MDH-- The above findings suggested that there might be previously unidentified sites in the respiratory chain that are upstream from and more sensitive to Zn²⁺ inhibition than the bc₁ complex. Possible sites of Zn²⁺ inhibition include mitochondrial dehydrogenases associated with the oxidation of the various metabolites tested above. Therefore, the Zn²⁺ sensitivities of the relevant mitochondrial dehydrogenases were directly tested.

Incubation of mitochondrial lysates with 100 µM Zn²⁺ resulted in a ~2-fold activation of SDH activity (data not shown). Purified mitochondrial MDH was unaffected by 100 µM Zn²⁺ (Fig. 3A), whereas purified GDH activity was inhibited by Zn²⁺ (Fig. 3B) with K_i^app = 6.1 ± 0.6 µM. In contrast, as detailed below, KGDHC activity was inhibited by submicromolar concentrations of Zn²⁺. The high Zn²⁺ sensitivities observed for both -KG-stimulated mitochondrial respiration and purified KGDHC activity (relative to the other substrates and dehydrogenases) supports the hypothesis that KGDHC is a target for intracellular Zn²⁺. To obtain additional evidence in support of this conclusion, detailed experiments delineating the effect of Zn²⁺ on purified KGDHC were carried out.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Effect of Zn2+ on MDH and GDH activities. Dixon plots of activities of purified MDH (~160 milliunits/ml; A) and GDH (~300 milliunits/ml; B) in the presence of varying concentrations of Zn2+.

Preparation of Chelator-free KGDHC-- Preliminary experiments with purified KGDHC indicated that Zn²⁺ in the range of 2-5 µM inhibited enzyme activity. However, the value of the determined K_i^app progressively declined as the concentration of enzyme in the reaction mixture was reduced (data not shown). The enzyme storage buffer contains EDTA and EGTA, both highly avid chelators of Zn²⁺ (52), and BSA, which has been reported to bind Zn²⁺ with submicromolar affinity (53). Direct dilution of KGDHC in storage buffer into assays results in concentrations of 2-5 µM for both EDTA and EGTA and 0.1-0.3 µM BSA in the final assay mixture. Therefore, the residual concentration of chelators was high enough to interfere with the determination of inhibition constants for metal ions. EDTA, EGTA, and BSA were simultaneously removed from the enzyme preparation by rapid gel filtration chromatography on a Sephadex 200 column. As expected, the apparent K_i^app for Zn²⁺ decreased after these chelators were removed from the enzyme stock. Denaturing gel electrophoretograms stained with Coomassie Blue confirmed that G-150 chromatography removed 97% of the BSA initially found in the enzyme preparation (data not shown). KGDHC subjected to gel filtration was used in all of the experiments presented below.

Inhibition of KGDHC Activity by Zn²⁺ Is Concentration-dependent-- Fig. 4A illustrates the inhibition of the KGDHC reaction by Zn²⁺ in the presence of saturating substrates and co-factors. Zn²⁺ addition to KGDHC results in a dose-dependent slowing in the rate of reaction product formation (Fig. 4A). The slow onset of inhibition causes a notable curvature in the reaction progress curves, presenting a dilemma in the assignment of velocity values used in the determination of K_i^app. In the experiments described below a standard interval of 15 min was arbitrarily chosen for calculating velocity (V_av). Dixon plots of 1/V_av versus the Zn²⁺ concentration for three different enzyme concentrations (Fig. 4B) resulted in similar intercepts on the x axis, providing an estimate of K_i^app (49). The lack of dependence of K_i^app on enzyme concentration indicates the successful removal of chelators. The average K_i^app for Zn²⁺ for four preparations of chelator and BSA-free KGDHC was 0.37 ± 0.05 µM.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   KGDHC inhibition by Zn2+ and disinhibition by EDTA. Chelator-free KGDHC was prepared and diluted into a standard reaction mixture, as described under "Materials and Methods." A, reaction progress curves for KGDHC in the presence of indicated concentrations of Zn2+ with no preincubation. Concentration of KGDHC was 4 µg/ml (~22 milliunits/ml). B, Dixon plot of Zn2+ inhibition at three input concentrations of KGDHC, as indicated. The values for Kiapp determined by least square fitting of the linear regression lines for 1, 2, and 4 µg/ml KGDHC are (mean ± S.E.) 0.19 ± 0.08, 0.22 ± 0.07, and 0.34 ± 0.06 µM, respectively. C, disinhibition by EDTA. Inhibition by residual divalent ions in the buffer preparation can be prevented by addition of submicromolar EDTA prior to assay. Plot of Vav versus EDTA concentration. The lines are independent linear regression fits to data points corresponding to 0-0.3 and 0.4-1.0 µM EDTA, respectively. The lines intersect at 0.3 µM Zn2+.

The sensitivity of KGDHC to inhibition by low concentrations of Zn²⁺ raised the possibility that KGDHC might be inhibited by residual Zn²⁺ or another metal in an assay mixture that is free of chelators. Fig. 4C demonstrates that V_av increases up to 2-fold upon addition of EDTA to the assay reaching a plateau at ~0.3 µM EDTA. The source of this metal has not been determined.

Inhibition of KGDHC Activity by Zn²⁺ Requires Enzyme Cycling-- The gradual onset of inhibition by Zn²⁺, reflected by curvature during the first minutes of the reaction (Fig. 4A), is consistent with a slow binding mechanism (54). Preincubation of KGDHC with Zn²⁺ might be expected to allow the slow binding step to take place prior to initiation of the enzyme reaction. However, preincubation of KGDHC with Zn²⁺ in the presence of all substrates except -KG for 78 min did not abolish the curvature of the reaction progress curves (data not shown). This observation indicates that substrate cycling in the presence of Zn²⁺ is required for the development of inhibition of KGDHC.

The requirement for substrate cycling in the inhibition of KGDHC is further illustrated by the data in Table II. Preincubation of KGDHC with Zn²⁺ for up to 78 min did not change the value of K_i^app when V_av was determined from the initial 15-min interval that followed addition of -KG. In contrast, K_i^app declined steadily when V_av was determined for intervals that were sampled after increasing periods of substrate cycling (Table II). These data indicate that after about 1 h of cycling the calculated K_i^app was reduced by about one half.

Partial Reversibility of Inhibition of KGDHC by Zn²⁺-- The KGDHC-catalyzed reactions represented in Fig. 4A were allowed to proceed in the presence of 0-5 µM Zn²⁺ for 110 min. At the end of this period, each reaction mixture was adjusted to a final concentration of 10 µM EDTA, and the reaction progress was again monitored (Fig. 5A). A gradual increase in enzyme activity was apparent. Control experiments indicated that 10 µM ETDA was sufficient to attain maximal reversal of KGDHC inhibition for Zn²⁺ concentrations up to 5 µM (data not shown). However, the extent of reversal of KGDHC inhibition was dependent upon the prior concentration of free Zn²⁺. This effect was manifested in two ways: the time to reach a linear rate of product formation was greater, and the maximal rate of product formation was lower for samples that were previously exposed to higher Zn²⁺ (e.g. compare 0.5 and 5 µM traces in Fig. 5A).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Reversal of Zn2+ inhibition is slow and depends upon prior Zn2+ exposure. A, dependence on prior Zn2+ concentration. Reactions similar to those in Fig. 4A were allowed to proceed for 49 min in the presence of the indicated concentrations of Zn2+. EDTA (0.2 mM, pH 8.0) was then added to a final concentration of 10 µM, and the reaction was monitored for 35 min. B, dependence on the duration of prior Zn2+ exposure. Each reaction was run in the presence of 1 µM Zn2+ for the times indicated. Reaction progress curves were recorded after addition of 10 µM EDTA.

The extent of KGDHC recovery was also dependent upon the duration of exposure to Zn²⁺. Fig. 5B shows the recovery for reaction mixtures that were exposed to the same concentration of Zn²⁺ (1 µM) for different reaction durations prior to reversal by EDTA. The time to reach maximum reaction velocity and the magnitude of this velocity after EDTA reversal diminished as the duration of exposure to a fixed Zn²⁺ increased.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Initial observations that Zn²⁺-inhibited mitochondrial respiration (32, 33) led to a series of studies that culminated in the proposal that the bc₁ complex of the electron transport chain is a target for Zn²⁺ (34, 35). However, Zn²⁺-induced inhibition of upstream dehydrogenases was not systematically examined (see the Introduction). The coincidence of elevated intracellular free Zn²⁺ and reduced carbohydrate flux that was reported in some disease states (16-21) led us to hypothesize that elevated intracellular Zn²⁺ directly inhibits NADH-producing dehydrogenases, in addition to inhibiting electron transport. This hypothesis was investigated by varying the concentration of Zn²⁺ within the physiological range (37) while monitoring respiration of isolated mitochondria. The effect of Zn²⁺ on the activity of selected mitochondrial dehydrogenases was also determined.

The changes in mitochondrial respiration that were induced by Zn²⁺ are unlikely to be due to changes in mitochondrial integrity. Zn²⁺ has been implicated in several mitochondrial changes, such as ion transport (55-57) and membrane swelling (33, 56, 58, 59). Membrane swelling has been associated with the opening of the mitochondrial permeability transition pore, loss of membrane potential, and uncoupling of respiration (60). However, Mg²⁺ was included in the respiration buffer. Control experiments have established that 3 mM Mg²⁺ prevents induction of the permeability transition by Zn²⁺.²

The absence of any absorbance change in mitochondrial suspensions also argues against Zn²⁺-induced disruption of mitochondrial membrane integrity.

Zn²⁺ Inhibits Mitochondrial Respiration Supported by -KG, Glutamate/Malate, or Succinate-- The order of sensitivity to Zn²⁺ inhibition among the various substrates tested in intact mitochondria was -KG > glutamate/malate > succinate (Figs. 1 and 2; Table I). Electrons (reducing equivalents) from glutamate/malate or -KG enter the electron transport chain as NADH at complex I. Electrons from succinate enter the electron transport chain as FADH₂ at complex II. Electrons from both complexes I and II then feed into the Q cycle portion of complex III (cytochrome bc₁ complex). It was reported that purified cytochrome bc₁ is inhibited by submicromolar concentrations of Zn²⁺ (34, 35). However, the greater Zn²⁺ sensitivity observed for intact mitochondria respiring on complex I substrates (-KG or glutamate/malate) than on complex II substrate (succinate) (Fig. 2) indicates that a site upstream from complex III is more sensitive to Zn²⁺-mediated inhibition, at least for intact mitochondria. The greater sensitivity of -KG stimulated respiration suggested that KGDHC might be the most Zn²⁺ sensitive upstream factor. We therefore compared the Zn²⁺ sensitivity of KGDHC to other dehydrogenases that oxidize mitochondrial substrates used in this study.

KGDHC Sensitivity to Zn²⁺ Inhibition-- The activity of purified KGDHC is potently inhibited by Zn²⁺ (Fig. 4), consistent with the high Zn²⁺ sensitivity of -KG-stimulated mitochondrial respiration. Purified GDH is ~15-fold less sensitive to Zn²⁺, and Zn²⁺ does not inhibit SDH and MDH activities. The weaker but appreciable inhibition of mitochondrial respiration observed in the presence of substrates other than -KG suggests that there may be other mitochondrial targets for Zn²⁺. One such possible target is the succinate transporter, which is inhibited by Zn²⁺ in bacteria (61). Also, some inhibition of complex I by Zn²⁺ cannot be excluded by our data.

Zn²⁺ Inhibition of KGDHC Is Time- and Activity- dependent-- Zn²⁺ inhibits KGDHC activity with a K_i^app of ~0.4 µM for the intact, purified enzyme (Fig. 4B) and K_i^app of ~1 µM for intact mitochondria respiring on -KG (Fig. 2C). Slow onset of Zn²⁺ inhibition was observed for the isolated enzyme in the presence of Zn²⁺ and -KG (Fig. 4A). Slow onset and slow reversal (Fig. 5A) of inhibition is often observed with tightly binding inhibitors (54). The values of K_i^app reported here represent an upper limit for the true K_i because of two considerations. First, Zn²⁺ inhibition increases with time as illustrated in Table II, but the determination of K_i^app was based on substrate formed during the first 15 min after initiation of the reaction. A second factor that may contribute to an excessively high estimate of the K_i for Zn²⁺ is the presence of ~0.3 µM endogenous Zn²⁺ or other chelatable divalent ion inhibitors within the KGDHC reaction mixture (Fig. 4C). Taken together, these considerations suggest that the actual K_i may be 0.1 µM or less.

The K_i^app for KGDHC and Zn²⁺ progressively diminishes with continued substrate cycling in the presence of -KG. In contrast, preincubation in the absence -KG has no effect on the value of K_i^app (Table II). Substrate cycling may be required because Zn²⁺ binds to a site on the enzyme that becomes available only during turnover. For example, Zn²⁺ might inhibit KGDHC activity by binding to the lipoyl prosthetic group of E2. The lipoyl group (i.e. [LipS₂]-E2) is oxidized in the resting state of the enzyme. Therefore, strong (bidentate) binding is only possible after complete reduction of the lipoyl group to a di-thiol (i.e. [Lip(SH)₂]-E2), which requires the presence of substrates (Equations 2 and 3). Similarly, E3 in the resting state contains a disulfide that is reduced to a dithiol by Lip(SH)₂-E2 (62), which is available only during enzyme cycling. If Zn²⁺ inhibition of E3 involves dithiol binding, then it necessarily requires enzyme cycling. Reduced E3 is only likely to exist when [Lip(SH)₂]-E2 is available, which is substrate-dependent. Other explanations for slow onset of inhibition are possible. For example, Zn²⁺ may form an inhibitory complex with one of the enzyme products (e.g. NADH; see below) generated during enzyme cycling. Preliminary experiments of the subunit reactions suggest that both E1/E2 and E3 activities are sensitive to Zn²⁺ inhibition. Further studies are needed to clarify the precise mechanism by which Zn²⁺ inhibits intact KGDHC.

Partial Reversibility of Zn²⁺ Inhibition of KGDHC-- The recovery of KGDHC activity is independent of EDTA concentration beyond that which is necessary to stoichiometrically bind Zn²⁺. This finding indicates that dissociation of Zn²⁺ from the inhibited complex is a unimolecular process followed by sequestration of free Zn²⁺ by EDTA. The dependence of recovery of KGDHC activity on both the dose and duration of exposure to Zn²⁺ suggests that initially the inhibited enzyme complex is fully reversible but that it is subsequently converted to a second irreversible or slowly reversible form. In the terminology of Morrison and Walsh (54), the first complex exhibits "slow" inhibition, whereas the second is characteristic of "slow-tight" inhibition. The gradual onset of Zn²⁺ inhibition and gradual recovery after addition of EDTA is characteristic of a slow binding complex. The development of stable inhibition characterized by a gradual loss of EDTA reversibility reflects the conversion from a slow to a slow-tight binding complex. The gradual development of inhibition is not simply a consequence of substrate consumption, because the fraction of substrate consumed is 5% or less.

Physiological Implications-- Our data demonstrate that concentrations of Zn²⁺ reported to occur in cultured hepatocytes (37) inhibit KGDHC activity. KGDHC activity within intact liver mitochondria is partly inhibited by 0.4 µM and completely inhibited by 3.2 µM Zn²⁺ (Fig. 1); an even lower range of Zn²⁺ concentrations (Fig. 4) inhibits purified KGDHC. The estimated range of cytosolic Zn²⁺ concentration in cultured hepatocytes (0.6-2.7 µM) (37) overlaps with the range of mitochondrial sensitivity observed in this report. The correspondence between the availability in cells and the sensitivity of mitochondria leads us to propose that modest fluctuations of intracellular Zn²⁺ may play a physiological role in the regulation of mitochondrial energy metabolism. Inhibition of KGDHC by chronic low doses of Zn²⁺ or high doses of short duration is readily reversible (Fig. 5). Therefore, mild or transient elevation of cellular free Zn²⁺ levels would be predicted to transiently reduce KGDHC-dependent respiration. More severe or prolonged exposure to elevated Zn²⁺ would result in irreversible inactivation of some or all of the available KGDHC and could lead to persistent inhibition of mitochondrial respiration. Preliminary experiments indicate that the mitochondrial pyruvate dehydrogenase complex can also be inhibited by micromolar concentrations of Zn²⁺. More precise definition of the potential role of Zn²⁺ as a physiological regulator of mitochondrial oxidative metabolism will require further studies.

Pathological Implications-- Changes in the distribution of free Zn²⁺ have been described for several disorders, including hypoxia/ischemia (2, 3) and Alzheimer's disease (63-65). Zn²⁺ influx into cells is associated with neuronal death (2, 3, 10). Moreover, direct involvement of Zn²⁺ in cytotoxic mitochondrial changes, including free radical formation, have been suggested (7, 8, 66).²

Oxidative energy metabolism is impaired in many neurodegenerative disorders (22-24). In particular, KGDHC activity in brain is reduced in Alzheimer's disease and a number of other neurodegenerative disorders (25, 26, 29, 67), but the possible relationship of the enzyme deficiency to Zn²⁺ levels in these conditions has not yet been explored. The decrease in KGDHC activity in Alzheimer's disease brain has been reported to exceed the decrease in KGDHC protein (67). Imbalances in the distribution of Zn²⁺ (63-65) in Alzheimer's disease brain may contribute to the loss of KDGHC activity.

The data in the present study indicate that damage because of pathological elevations of Zn²⁺ may occur, at least in part, through inhibition of -KG oxidation at the KGDHC-catalyzed step. As discussed above, shorter exposure to relatively lower levels of Zn²⁺ is associated with reversible inhibition of KGDHC, and low levels of Zn²⁺ may thus play a role in metabolic regulation. However, prolonged exposure to higher concentrations of Zn²⁺ may lead to slow and incomplete recovery from inhibition of KGDHC activity and mitochondrial oxidative function (Fig. 5). The present findings raise the possibility that extreme elevations of Zn²⁺ that may exist under pathological conditions lead to such severe and long-lasting inhibition of -KG oxidation in mitochondria that they contribute to cell death. This possibility also needs to be tested by further experiments.

Conclusion-- The data reported here raise the possibility of a previously unsuspected role of Zn²⁺ in the normal regulation of metabolism, namely, at the KGDHC-catalyzed step of mitochondrial oxidation through the Krebs tricarboxylic acid cycle. Oxidative energy metabolism is impaired in many neurodegenerative disorders and heart disease, particularly at the step catalyzed by KGDHC. The current studies indicate that Zn²⁺ may well play a critical role in these disorders. Further studies of the role of Zn²⁺ in the regulation of energy metabolism in health and disease are warranted.

	FOOTNOTES

^* This work was supported by National Institutes of Health (NIH)/NINDS Grant NS38741 (to A. M. B.). Pilot support was provided from Leadership and Excellence Award in Alzheimer's Disease AG09014 NIH/NIA (to J. P. B.) and the Winifred Masterson Burke Relief Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dementia Research Service, Burke Medical Research Inst., 785 Mamaroneck Ave., White Plains, NY 10605. Tel.: 914-597-2361; Fax: 914-597-2757; E-mail: abrown@burke.org.

Deceased March 2, 1999.

² B. S. Kristal and A. M. Brown, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: KGDHC, -ketoglutarate dehydrogenase complex; -KG, -ketoglutarate; BSA, bovine serum albumin; CoA, coenzyme A; CoASH, coenzyme A, reduced form; DTT, dithiothreitol; E1, -ketoglutarate dehydrogenase; E2, dihydrolipoyl acyltransferase; E3, dihydrolipoyl dehydrogenase (lipoamide dehydrogenase); GDH, glutamate dehydrogenase; MDH, malate dehydrogenase; LipS₂, lipoamide, oxidized form; Lip[SH]₂, reduced lipoamide (=dihydrolipoamide); SDH, succinate dehydrogenase; TPP, thiamin pyrophosphate.

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